Saliva is the principal protective agent for the mouth and thus is of primary importance to oral health maintenance. Perturbations of salivary secretory mechanisms can consequently lead to serious oral health problems. The objective of this project is to study the membrane and cellular processes that underlie the phenomenon of salivary fluid secretion and thus to contribute to our understanding of the fluid secretory process. Because similar secretory mechanisms are thought to be common to a number of other tissues, this information should be of rather broad applicability and interest. During the present reporting period we have continued our in-depth studies of the salivary Na-K-2Cl cotransporter (NKCC1). We have also continued our work on the structural and functional properties of presenilin 1, the putative proteolytic component of gamma-secretase. NKCC1 is thought to be the major Cl entry pathway into salivary acinar cells and thus to be primarily responsible for driving Cl secretion, and thereby fluid secretion, in salivary glands. Obtaining a better understanding of the structure/function relationships of this protein and its behavior in acinar cells will improve our knowledge of salivary function and dysfunction, as well as possibly providing indications of how to treat the latter. In past studies we have established that the functional unit of NKCC1 is a homodimer. During this reporting period we have shown that the region of NKCC1 responsible for this dimerization lies in its intracellular C-terminus. In subsequent experiments in which we replaced the C-terminus of NKCC1 with the C-termini of several closely related transporters from the same gene family we found that these chimeric proteins formed homodimers but not heterodimers. These results are consistent with the hypothesis that all members of this gene family exist in a dimeric state but that their C-terminal dimerization motifs are quite specific so that heterodimers between different family members do not occur. Additional experiments to identify the amino acids involved in NKCC1 dimerization are now underway. Mutations in presenilin 1 (PS1) have been linked to cases of familial early-onset Alzheimer's disease. This protein is thought to be the proteolytic component of gamma-secretase, the protease that is responsible for the intramembrane cleavage of a number of substrates including the beta-amyloid precursor protein, the protein that is primarily responsible for the senile plaques characteristic of Alzheimer's disease. During the present reporting period we have continued our experiments examining the transmembrane topology of PS1. We have now obtained convincing results from three independent methods, as well as from functional studies, that the C-terminus of PS1 is located in the extracellular compartment. This is a particularly interesting result since it indicates that a region of PS1 near its C-terminus previously identified as involved in substrate binding is located within the membrane as opposed to residing in the intracellular compartment as was formerly thought. The functional significance of this observation is now under investigation. Considerable evidence indicates that PS1 is involved in intracellular calcium signaling. Specifically it has been found that the expression of Alzheimer?s disease-associated PS1 mutants results in enhanced accumulation of calcium in intracellular stores and larger agonist-induced calcium transients. It has been suggested that this effect is associated with increased gamma-secretase activity. We have used various inhibitors to block gamma-secretase activity in the human submandibular cell line HSG. We expected that these inhibitors would have the opposite effect of PS1 mutants, i.e., that they would reduce the calcium content of intracellular stores and blunt agonist-induced calcium transients. Surprisingly, however, we found that most of these inhibitors hadd little or no effect on carbachol-induced calcium mobilization or store content in HSG cells, and that those inhibitors that did blunt calcium mobilization did so by a mechanism apparently unrelated to gamma-secretase activity. These results indicate that the effects of PS1 on calcium signaling may be more complex than previously thought and may not be related directly to gamma-secretase activity.